Methods of treating cancer using flavonoid glycosides and steroidal glycosides

ABSTRACT

The invention relates to the use of flavonoid glycosides and steroidal glycosides from Hosta or their derivatives as functional ingredients in functional food, OTC and pharmaceutical composition to prevent or treat cancer. The invention further includes methods for making and methods for using the invention.

[0001] This application is a continuation-in-part of application Ser.No. 09/578,649, filed May 25, 2000, the entire disclosure of which isincorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention relates to the use of novel flavonoid and steroidalglycosidic compounds from HOSTA for preventing and treating cancer. Theinvention further includes methods for making and methods for using theinvention.

BACKGROUND OF THE INVENTION

[0003] Since ancient times a vast number of natural remedies of plantand animal origin has been used for medical treatment and diseaseprevention. The famous Chinese medicine Encyclopaedia “Ben Cao Gang Mu”(52 Volumes) was written by Li Shih Zhen and published in 1596 [1]. Thisbook described 1892 drugs shape, character, taste, source, method ofcollection and preparation and treatment, prevention of various disease.

[0004] A fast growing body of evidence obtained in the recent years byutilization of modem scientific, experimental and clinical methodsconfirms the biological activity of many micro components of plants thatcan be utilized in prevention or treatment of a variety of chronicdiseases, including cancer and cardiovascular disease [2-4].

[0005] Despite the fact that scientific evaluation of medicinal plantshistorically has been responsible for discovery of a multitude of modernmedicine, approximately only 1% of plants has been analyzed so far.

[0006] The analysis of botanical material is not a trivial matter.Usually, a sample to be analyzed contains a very complex mixture of manycomponents. Only some of them might be biologically active, while othermay be toxic. Components of these complex mixtures are usuallyinteracting amongst themselves . Frequently, in many plants, dozensof-species and strains of the same genus, differ substantially incontent of the active ingredients. Even within the same plant, differentparts often have different chemical composition. Furthermore, thepresence and concentration of some substances depend greatly on thesoil, location, season, time of harvest, storage conditions, handlingmethods, conditions and solvents used for extraction, etc. Thisdiversity of important conditions affecting the quality of botanicalremedies requires therefore implementation of stringent, well-designedand closely-monitored standard operating procedures of manufacturing toensure consistency from batch to batch of a nutraceutical product,followed by application of an appropriate analysis to ensure consistentpotency and efficacy.

[0007] The major aims of qualitative analyses in phytochemistry includemonitoring of the preparative isolation and purification ofphytochemicals, chemotaxonomic testing and drug identification and/ordetection of adulterants [5-6].

[0008] Plant constituents often exist in the form of glycosides. Theseconjugates may or may not occur together with their respectiveaglycones. Many glycosides play an important role as drugs and dyes.Glycosides are thermally labile, polar and non-volatile compoundsfrequently differing in their solubility and biological activity fromtheir respective aglycones. By changing a type and/or number of attachedsaccharides the physicochemical and biological properties of theglycosides can be modified [7].

[0009] Among phytochemicals existing in the glycosilated form thatdeserve a special attention due to their wide distribution in nature anda high number of beneficial biological and medicinal properties, aresaponins and flavonoid glycosides. Saponins are high molecular weightglycosides consisting of a sugar moiety linked to triterpine or steroidaglycones [8]. The most common sugars encountered in saponins arehexoses (glucose, galactose and mannose), 6-deoxyhexoses (rhamnose),pentose (arabinose and xylose), uronic acids (glucuronic acid andgalacturonic acid) or amino sugars (glucosamine and galactosamine).Sugars may be linked to the sapogenin at one or two glycosylation sites(through an ether or/and an ester linkage), giving the correspondingmonodesmodic or bidesmosidic saponins, respectively [9-10].

[0010] Because of the glycosylation of their hydrophobic aglycones,saponins act as biological detergents and, when agitated with water,form a soapy lather that gives rise to name of this group of compound[8]. From a biological point of view saponins have diverse groupproperties, some deleterious, but many beneficial. Some saponins havebeen used as plant drugs in folk medicine. They may exhibit cardiacactivity, hemolytic activity, activity as fish poisons,hypocholesterolemic [11] immunostimulatory and anti-tumorigenic activity[8]. They can be used as bitterness and sweetness modifiers,allelochemicals and cosmetic ingredients. The second important classesof phytochemicals, which attracted a high interest due to its widedistribution in nature and diversified biological properties areflavonoids [12-13]. These polyphenolic compounds, apart from catechinsand proanthocyanidins, consist mainly of glycosides of flavonols,flavons, flavanones, anthocyanins and less frequently isoflavons or freeaglycones. Flavonoids represent an important constituent of many edibleplants and are present in foods and beverages derived from plants.

[0011] Some flavonoid-containing species have been used in traditionalmedicine. Recently these phytomedicines have been extensivelyinvestigated and their health benefits confirmed in many cases for thelong-term treatment of mild and chronic diseases or in attaining andmaintaining a condition of well-being. Flavonoids function as strongantioxidants [3], free-radical scavengers, and metal chelators and theirbiological properties can also be linked with their interaction withenzymes, adenosine receptors, and biomembranes [14-15]. Many of thebioflavonoids exhibit very beneficial pharmacological activities, suchas anti-inflammatory, antiallergic, antimicrobial, antioxidative,enzyme-inhibitory effects, and etc. [3]

[0012] The identification of individual flavonoids, sapogenins and theirglycosides has long been carried out by Mass Spectrometry [8, 12, 16],Ultraviolet Spectroscopy [8, 12, 17] and 13C-NMR [8, 18]. Thesetechniques were executed on highly purified compounds and were notapplied to mixtures.

[0013] Separation of individual flavonoids, sapogenins and theirglycosides from each other has long been carried out by Paper, ThinLayer and Open Column Chromatography [8, 12].

[0014] More recently HPLC has been used for the separation of individualflavonoids, sapogenins and their glycosides from each other [8, 12 ].These techniques gave limited resolution between individual glycosidesof either the flavonoids or the sapogenins.

[0015] The combination of HPLC and Diode Array UV-Visible Detection gavenew possibilities in qualitative analysis of flavonoids in plantextracts [19-20]. Information about the type and number of glycosidicunits was lost due to the preparation. The extraction and purificationdid not address the identification and quantization of individualglycosides. Mass Spectrometry with thermospray Ionization permittedroutine online analysis of a number of glycosides of both flavonoid andsapogenin classes, but sensitivity was limited and interpretation wascomplicated by the frequent formation of artifacts [21-22]. Moreover,due to the relatively energetic ionization of the thermospray techniquethe higher glycosides were not observed. Continuous Flow Fast AtomBombardment gave some advantages in ionization of small polar moleculesbut at the cost of instrumental complexity and reliability [23].

[0016] The advent of Electrospray Ionization permitted molecules to beionized with very low energies under atmospheric pressures and at roomtemperatures. Very polar, high molecular weight species could beroutinely analyzed with little artifact formation that could complicateinterpretation [24-25]. The technique also permits the use of CollisionInduced Fragmentation for generating ions that aids in structureelucidation [26].

[0017] The combination of three powerful techniques LC/DAD/ESIMS wasused to study the aglycones and glycosides present in berries [24].These works however largely concentrated on the identification of theflavonoid aglycones or of glycosides of no greater than two units.

[0018] The present invention provides a fast and reliable method for thesimultaneous analysis of both flavonoid glycosides and steroidalglycosides in one procedure. As a model to show the usefulness of thistechnique we have chosen plants from Hosta genus which belongs to thesubfamily Asphodeloideae in Liliaceae. These plants are widelydistributed thus offering easy and economical access to this source offlavonoid and steroidal glycosides of potential medicinal application.The flowers, leaves and rhizomata of hosta have been used as a folkmedicine in China [1, 27]. A steroidal saponin identified ashexasaccharide and prepared from the extract of dried Hosta leaves by O.Masamitsu, et al. [28-29] exhibit antibacterial and antitumor activitywhile some of the steroidal glycosides identified by M. Mimaki groupdisplayed cytostatic activity on HL-60 cells.

[0019] Although eight kaempferol glycosides [30] and twenty sixsteroidal glycosides [28-29, 31-37] have been previously separated fromHosta leaves and Hosta rhizomers, respectively, there has been noreports of any comprehensive procedure to extract simultaneously bothclasses of glycosides from the Hosta leaves.

SUMMARY OF THE INVENTION

[0020] The present invention is based on the discovery of novelflavonoid glycosides and steroidal glycosides from Hosta that exhibitanti-cancer activities. The present invention provides a method toprevent or treat susceptible cancers in humans comprising administeringa cancer-treating amount of these compounds. Preferably, the presentmethod will be utilized to treat or prevent chronic myelogenousleukemia, liver cancer and lung cancer. Another aspect of the presentinvention is a method as disclosed which utilizes oral or intravenousadministration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a graph illustrating details of the instrumental setup.

[0022]FIG. 2 is a graph showing an application of Collisionally InducedDissociation for the identification of sugars from a steroidalhexaglycoside where the steroidal aglycone corresponds to an open spiroring sapogenin and the losses of m/e 162 fragment indicates the presenceof five hexose (most likely glucose) moieties and the loss of m/e 146fragment indicates the presence of one 6- deoxyhexose (most likelyα-L-rhamnose).

[0023]FIG. 3 is a graph showing an application of Collisionally InducedDissociation for the identification of sugars from a flavonoidtetraglycoside where the flavonoid aglycone corresponds to kaempferoland the loss of m/e 162 fragment indicates the presence of hexose (mostlikely glucose).

[0024]FIG. 4 is a graph depicting chemical structures of eightkaempferol glycosides, previously found in the extract of Hosta leaves.

[0025]FIG. 5 is a graph depicting chemical structures of four steroidalglycosides, previously found in the extract of Hosta rhizomers.

[0026]FIG. 6 is a table containing a list of all flavonoid and steroidalglycosides from the Hosta leaves extracts found by the application ofthis invention, and compared with glycosides previously identified inthe literature.

[0027]FIG. 7 is a table containing a list of eight new glycosidiccompounds identified for the first time in the Hosta leaves extracts byapplication of the presented procedure.

[0028]FIG. 8 is a graph that summarizes the procedure used forextraction, pre-purification and analyses of Hosta leaves in Example 1.

[0029]FIG. 9 is a LC/MS chromatogram of the raw extract from Example 1.

[0030]FIG. 10 is a graph that summarizes the procedure used forextraction, prepurification and analyses of Hosta leaves in Example 2.

[0031]FIG. 11 is a LC/MS chromatogram of a pre-purified mixture offlavonoid and steroidal glycosides extract from Example 2.

[0032]FIG. 12 is a graph which summarizes the procedure used forextraction, prepurification and analyses of Hosta leaves in Example 3

[0033]FIG. 13 is a LC/MS chromatogram of the raw extract from Example 3.

[0034]FIG. 14 is the result of a cell assay that showed specificanti-cancer activity of S1, an extract containing primarily a steroidalglycosides of molecular weight 1,406 Da. This sample exhibitsconsiderable anti-proliferation activity against three cell lines fromCML, liver, and lung cancer.

[0035]FIG. 15 is the result of a cell assay that showed specificanti-cancer activity of F2-2, a flavonoid glycoside mixture exhibitedselective activity on inhibition of a lung cancer cell line

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0036] The present invention is based on the discovery of novelflavonoid glycosides and steroidal glycosides from Hosta that exhibitanti-cancer activities. The present invention is applicable, but notlimited, to the extraction, isolation and simultaneous determination ofboth flavonoid and steroidal glycosides from plants, fresh or dried, andother potential natural sources of nutraceuticals. This procedure isalso fully applicable to qualitative and quantitative analysis ofdifferent forms of herbal supplementations, such as powders, tinctures,suspensions, solutions, syrups, capsules, tablets, etc.

[0037] The biological material, such as plant, should be collected andstored under properly designed and controlled conditions, to ensure theconsistency in the content of active components. Measures should beapplied to avoid the presence of any harmful contaminants, such aspesticides, herbicides, heavy metals, etc. If drying is recommended, thechemical and enzymatic sensitivity of the active components should beconsidered. Thus the avoidance of exposure to light, elevatedtemperatures, oxygen presence or prolonged storage in aqueous solutionprone to facilitate biochemical degradation should be considered andapplied, if necessary.

[0038] Prior to the extraction procedure sample should be prepared bychopping into small pieces, blending, grounding or crushing, in order toimprove the contact of solvent with the extracted matter.

[0039] In the present invention, for analytical purposes, the extractionof the polar phytochemicals such as bioflavonoids or saponins from theplant or marine matter or their formulations is achieved by theapplication of water or aqueous solutions of a variety of polar solventssuch as lower alcohols, ketones, or acetonitrile. The elevatedtemperature and repeated extraction procedures might be used to improvethe extraction effectiveness. The effectiveness can be further enhancedby stirring, shaking or sonication.

[0040] Plant or marine matter consist of a multitude of components, bothpolar and nonpolar. In order to provide a simple, sensitive and reliablemethod for analyzing for phytochemicals, such as flavonoid and steroidalglycosides, the sample should be treated to remove contaminants andother undesired components that would interfere with the analysis. Sucha removal, for analytical purposes, can be achieved by precipitation ofsome undesired compounds by means of concentration of the extract volumeand/or refrigeration.

[0041] Further removal of interfering less polar compounds can beachieved by subjecting the crude extract to liquid-liquid extractionwith a water immiscible organic solvent, such as alkanes, cycloalkanes,ethers or lower esters.

[0042] The purification of sample from polar components such asinorganic salts, simple sugars, and aminoacids that could interfere withthe final analysis of the phytochemicals of interest can be achieved,for analytical purposes, by application of open column or flash columnchromatography using a variety of different stationary phases, such aspolyamide resin or a weakly acidic cation exchange resin, such asAmberlite IRC-50, and mixture of water and lower alcohols as mobilephase.

[0043] Such separation can be easily monitored by HPLC or TLC with avariety of detection methods. The column chromatography may also utilizeother modes, such as normal phase chromatography with application ofsilica gel or alumina or gel filtration approach.

[0044] Thus such extracted and pre-purified sample can be then subjectedto qualitative and quantitative analysis by the application of reversephase HPLC in an isocratic or preferably gradient mode. The combinedapplication of two independent and powerful detection techniques ofElectrospray Mass Spectrometry and Diode Array Spectroscopy allow forthe selective and simultaneous identification of the individualcomponents, such as phytochemicals of interest, contained in thepre-purified extract. The application of Electrospray Mass Spectrometrydetection for the thermally liable compounds prevents creation ofartifacts that may lead to misinterpretation of the instrumental datathat was frequently possible with the previously used ionizationtechniques. It was found that the application of negative ion modeyields patterns that are more informative than other techniques,decreasing at the same time the risk of the artifact formation and datamisinterpretation. Furthermore, the post-column application oftriethylamine enhances the sensitivity of this method of detection. FIG.1 details the instrumental setup.

[0045] The sample preparation combined with the applied detection systempresented in this invention yield sensitive and extensive qualitativeinformation about the individual components of the analyzed extract,such as, for example flavonoid and steroidal glycosides. Thisinformation includes, but is not limited, to molecular weight, numberand type of glycoside substituents, and from the Diode ArraySpectroscopy absorption patterns, this technique allows fordifferentiation amongst different types of aglycones present in thecomponents of the extract.

[0046] The application of the presented procedure in combination withthe use of Collisionally Induced Dissociation can produce mass spectralpatterns that can allow structural data to be deduced. Those skilled inthe art will recognize that Mile 162 fragment could be related to a lossof hexose (e.g. glucose), that of m/e 146 fragment from the loss of6-deoxyhexoses (e.g. rhamnose), and that of m/e 132 fragment from theloss of pentose (e.g. xylose) (see FIG. 2 and FIG. 3).

[0047] As a very educational and powerful example for the application ofthis invention can serve the application of the present procedure forthe extraction, purification and analysis of flavonoid and steroidalglycosides from Hosta leaves. Thus this invention has allowed for thefirst time simultaneously identify a total of twenty glycosides, bothfrom the flavonoid and steroidal class (Table 1, FIG. 4). Among theidentified glycosidic compounds were all eight kaempferol glycosides,previously reported by J. Budzianowski (structures of which are shown inFIG. 5), and 4 steroidal glycosides, previously reported in theliterature by the Japanese researchers for the extracts of Hostarhizomers (structures of which are shown in FIG. 6). This procedure,with application of Collisionaly Induced Dissociation also allowed forthe identification of eight new, previously not detected in the Hostaplant extracts clycosidic compounds, seven of which were identified assteroidal tetra, penta, and hexaglycosides, and one as kaempferoltetraglycoside (Table 2, FIG. 7).

[0048] These novel compounds can be used for treatment of cancer.Particularly, the steroidal glycosides can be used to treat chronicmyelogenous leukemia, liver, and lung cancer since these compoundsshowed anti-proliferation activity against cell lines from thesediseases (FIG. 14). The flavonoid glycoside mixture F2-2, and theindividual compounds contained in it, can be used to treat lung canceras shown in FIG. 15. Treatment in accordance with the present inventioncan be accomplished by oral or intravenous administration along with apharmaceutically acceptable carrier such as rice syrup solids,maltodextrin, and hydroxypropylcellulose, or in a food, to a patienthaving cancer. Orally-administrable dosage forms of the invention mayinclude, but are not limited to, capsules, tablets, powders and liquids.The amount of active ingredient to be administered may vary dependingupon the type of cancer and the body weight of the individual beingtreated. Dosages of the active ingredient may be in the range of 1 mg/kgper day to 30 mg/kg/day. These dosages should be continued until nodetection of cancer is determined by standard means.

[0049] Another aspect of the present invention is a method as disclosedwhich utilizes oral or intravenous administration. However, those in theart will recognize that many avenues of administration are possible. Forinstance, administration of drug may be via capsule, tablet, solution,sachet, suspension, intravenously, orally, intramuscularly, includingimplantation into the tumor itself, topically or parenterally.

[0050] The present invention is believed to be utilizable with othertypes of cancers other than those described herein as would be known tothose skilled in the art. The application of the presented method ofextraction, purification and separation can be easily adopted to apreparation of pre-purified and standardized mixtures of phytochemicalsor the individual compounds in a pure form for additional structuralelucidation and/or conformation (e.g., by means of NMR spectroscopy orX-ray analysis) as well as for their screening for biological activity.

[0051] The presented invention does not require any additional steps,such as chemical derivation. However, it can be combined with either ananalysis of partly and/or fully hydrolyzed material, as well asconsecutive derivation of glycosides, and subjecting these samples tofurther HPLC/MSD/DAD or other methods of analysis. Applications ofappropriate standards allow for an easy, sensitive and highly reliablemethod of quantitative analysis, and therefore, can be widely utilizedfor standardization of nutraceuticals.

[0052] Thus the presented procedure represents a much more industriallyadvantageous method for the execution of these analyses, particularly inthe field of research, standardization and quality control of herbal andmarine matter, or any of their nutraceutical supplement formulations.

EXAMPLE 1

[0053] The procedure for extraction, pre-purification and analysis ofHosta leaves is depicted in FIG. 8. The fresh Hosta leaves (GoldenTiara) were hand picked in October. Fresh leaves (20 g) were choppedinto small pieces and then ground in a blender with water (500 mL),followed by sonication for 2 hours at 50° C. The extract was filtratedto separate and remove fibrous material. The extraction of the separatedsolid material was then repeated with another portion of water (500 mL).Both extracts were combined and concentrated to ca. 25 mL under reducedpressure on a Rotovapor. The concentrated aqueous solution was extractedtwice with hexane (25 mL) followed by two-time extraction with ethylacetate (25 mL). The aqueous phase was evaporated under vacuum todryness to afford 0.28 g (1.4% yield) of the raw extract of flavonoidand steroidal glycosides in the form of powder. This powder (0.28 g) wasdissolved in 3.0 mL of a mixture of water and ethanol (1:1) andsubjected to HPLC/MS analysis.

[0054] The solution of this raw extract was separated by a columnchromatography on Amberlite IRC-50 resin (16-50 mesh, Sigma Company)with gradient elution of increasingly higher content of ethanol inwater. The chromatography was monitored by HPLC/MSD/DAD. The firstfraction eluted with water (500 mL) contained mainly some polarinterfering compounds, such as sugars; the evaporation under vacuum todryness yielded a solid powder (110 mg). The second fraction was elutedwith 5% ethanol-water solution (200 mL). The third fraction was elutedwith 10% ethanol-water solution (200 mL) and the fourth fraction elutedwith 20% ethanol-water solution (200 mL). The second, third and fourthfractions were combined and evaporated under vacuum to dryness to afford48 mg (0.24% yield) of a crude mixture of flavonoid glycosides. Thefifth fraction was eluted with 50% ethanol-water solution (200 mL). Thisfraction was evaporated separately under vacuum to dryness to afford 28mg (0.14% yield) of a crude mixture of steroidal glycosides.

[0055] The HPLC/MSD/DAD analysis was performed with a system thatconsisted of an HPLC 1100 series LC/MSD (Hewlett-Packard) instrument,autoinjector, quaternary pump with on-line vacuum degassing unit,thermostated column compartment and diode array detector. At the sametime, a mass detector was used. The API-EI mode was chosen. The negativeion mode provided better sensitivity and the interpretation of thespectra was found to be easier. So the analysis results were obtained innegative mode at fragmentation potential of 100 cV. A standard Zorbax C8column (150 mm long×2.1 mm I.D.) with 5 μm particle size was used inthese examples.

[0056] Operation conditions for the analysis were as follows:

[0057] Temperature 30° C.

[0058] Mobile phase consisted of an ACN/water mixture gradient:

[0059] 0-6 minutes ACN 15%,

[0060] 6-18 minutes ACN from 15% to 90%

[0061] 18-20 minutes ACN from 90% to 15%

[0062] The mobile phase flow rate was 0.4 mL/min.

[0063] Wavelength of UV detector was recorded on 280 nm.

[0064] The mass ion scan was from 100 to 1800.

[0065] The recorded LC/MS chromatogram of the raw extract is presentedin FIG. 9.

EXAMPLE 2

[0066] The procedure for extraction, pre-purification and analysis ofHosta leaves is depicted in FIG. 10. The fresh Hosta leaves (Lemon Lime)were hand picked in September. Fresh leaves (150 g) were chopped intosmall pieces and then ground in a blender with 50% aqueous ethanolsolution (500 mL), followed by sonication for 2 hours at 50° C. Theextract was filtrated to separate and remove fibrous material. Theextract was concentrated to ca. 50 mL under reduced pressure on aRotovapor. The concentrated aqueous solution was diluted to 300 mL withwater, refrigerated overnight and the formed precipitate of undesiredcomponents such as alkylphenols and fat was filtered out. The resultedfiltrate was extracted twice with hexane (25 mL) followed by two timeextraction with ethyl acetate (25 mL). The aqueous phase was evaporatedunder vacuum to dryness to afford 2.7 g (1.8% yield) of the raw extractof flavonoid and steroidal glycosides in the form of powder. This powder(0.5 g) was dissolved in 5.0 mL of a mixture of water and ethanol (1:1)and subjected to chromatographic separation. The separation wasperformed by a column chromatography on Amberlite IRC-50 resin (16-50mesh, Sigma Company) with gradient elution of increasingly highercontent of ethanol in water. The chromatography was monitored by HPLC/UVand the analysis of the final combined fractions by HPLC/MSD/DAD. Thefirst fraction eluted with water (500 mL) contained mainly some polarinterfering compounds, such as sugars; the evaporation under vacuum todryness yielded a solid powder (260 mg). The second fraction was elutedwith 5% ethanol-water solution (150 mL). The third fraction was elutedwith 10% ethanol-water solution (150 mL) and the fourth fraction elutedwith 20% ethanol-water solution (150 mL). The second, third and fourthfractions were combined and evaporated under vacuum to dryness to afford45 mg (0.16% yield) of a crude mixture of flavonoid glycosides. Thefifth fraction was eluted with 50% ethanol-water solution (150 mL) andthe sixth one with 80% ethanol-water solution (200 mL). Fractions fiveand six were combined and evaporated separately under vacuum to drynessto afford 40.5 mg (0.145% yield) of a crude mixture of steroidalglycosides.

[0067] The HPLC/MSD/DAD analysis was performed with a system thatconsisted of an HPLC 1100 series LC/MSD (Hewlett-Packard) instrument,autoinjector, quaternary pump with on-line vacuum degassing unit,thermostated column compartment and diode array detector. At the sametime, a mass detector was used. The API-EI mode was chosen. The negativeion mode provided better sensitivity and the interpretation of thespectra was found to be easier. So the analysis results were obtained innegative mode at fragmentation potential of 400 eV. A standard Zorbax C8column (150 mm long×2.1 mm I.D.) with 5 μm particle size was used inthese examples. Operation conditions for the analysis were as follows:Temperature 30° C. Mobile phase consisted of an ACN/water mixturegradient:

[0068] 0-15 minutes ACN 15%

[0069] 15-20 minutes ACN from 15% to 90%

[0070] 20-25 minutes ACN 90%

[0071] 25-30 minutes ACN from 90% to 15%

[0072] The mobile phase flow rate was 0.4 mL/min.

[0073] Wavelength of UV detector was recorded on 280 nm.

[0074] The mass ion scan was from 100 to 1800.

[0075] The recorded LC/MS chromatogram of the pre-purified extract ispresented in FIG.

EXAMPLE 3

[0076] The procedure for extraction, pre-purification and analysis ofHosta leaves is depicted in FIG. 12. The fresh Hosta leaves (BlueDimples) were hand picked in September. Fresh leaves (40 g) were choppedinto small pieces and then ground in a blender with 50% acetonitrilesolution in water (250 mL), followed by sonication for 2 hours at 20-40°C. The extract was filtrated to separate and remove fibrous material.The extract was concentrated to ca. 5 mL under reduced pressure on aRotovapor. The concentrated solution was extracted twice with hexane (3mL) followed by two-time extraction with ethyl acetate (3 mL). Theaqueous phase was evaporated under vacuum to dryness to afford 1.8 g(4.5% yield) of the raw extract of flavonoid and steroidal glycosides inthe form of powder. This powder (1.8 g) was dissolved in 10.0 mL of amixture of water and acetonitrile (1:1) and subjected to HPLC/MSanalysis.

[0077] The solution of this raw extract was separated by a columnchromatography on polyamide resin (25 g, 80 mesh) with gradient elutionof increasingly higher content of ethanol in water. The chromatographywas monitored by HPLC/MSD/DAD. The first fraction eluted with water (500mL) contained mainly some polar interfering compounds, such as sugars;the evaporation under vacuum to dryness yielded a solid powder (1273mg). The second fraction was eluted with 5% ethanol-water solution (200mL). The third fraction was eluted with 10% ethanol-water solution (200mL) and the fourth fraction eluted with 20% ethanol-water solution (200mL). The second, third and fourth fractions were combined and evaporatedunder vacuum to dryness to afford 80.75 mg (0.2% yield) of a crudemixture of flavonoid glycosides. The fifth fraction was eluted with 50%ethanol-water solution (250 mL). This fraction was evaporated separatelyunder vacuum to dryness to afford 252 mg (0.63% yield) of a crudemixture of steroidal glycosides.

[0078] The HPLC/MSD/DAD analysis was performed with a system thatconsisted of an HPLC 1100 series LC/MSD (Hewlett-Packard) instrument,autoinjector, quaternary pump with on-line vacuum degassing unit,thermostated column compartment and diode array detector. At the sametime, a mass detector was used. The API-EI mode was chosen. The negativeion mode provided better sensitivity and the interpretation of thespectra was found to be easier. So the analysis results were obtained innegative mode at fragmentation potential of 100 eV. A standard Zorbax C8column (150 mm long×2.1 mm I.D.) with 5 μm particle size was used inthese examples.

[0079] Operation conditions for the analysis were as follows:Temperature 30° C.

[0080] Mobile phase consisted of an ACN/water mixture gradient:

[0081] 0-6 minutes ACN 15%

[0082] 6-18 minutes ACN from 15% to 90%

[0083] 18-20 minutes ACN from 90% to 15%

[0084] The mobile phase flow rate was 0.4 ml/min.

[0085] Wavelength of UV detector was recorded on 280 nm.

[0086] The mass ion scan was from 100 to 1800.

[0087] The recorded LC/MS chromatogram of the raw extract is presentedin FIG. 13.

EXAMPLE 4

[0088] These novel compounds were tested against seven cancer cell linesfor anti-tumor activity. The assays were performed with extractsobtained one step prior to the final purification of individualcompounds. The percentage of each component in the extracts wascharacterized. The anti-cancer activity of these compounds was examinedover a wide concentration range between 100 ug/mL to 0.191 ng/mL. S1,the extract containing five steroidal glycosides wherein 53.8% is acompound of molecular weight of 1,406 Da, exhibits considerableanti-proliferation activity against three cell lines from CML, liver,and lung cancer (FIG. 14). One of the flavonoid glycoside mixture, F2-2,showed selective activity on inhibition of the lung cancer cell lineA549 (FIG. 15). This mixture contains five flavonoid glycosides ofmolecular weight of 756, 918, 888, 726, and 902 Dalton respectively. Therelative amount for each compound is 12.6, 39.9, 24.2, 14.5, 4.8, and3.8%, respectively.

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Foreign Patent Documents

[0093] Ochi, M., et al., Steroid Saponin from Hosta and Antimicrobialand Antitumor Agents Containing It. JP 10 114,791 [98 114,791] (C1.C07J71/00), May 6, 1998, Appl. 96/270,292, Oct. 11, 1996; 12 pp; CA1129: 32293w

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What is claimed is:
 1. A method of preventing or treating cancer byadministering to a patient a therapeutically effective amount of asteroidal glycoside or mixtures thereof.
 2. The method of claim 1wherein the therapeutically effective amount of the steroidal glycosideis approximately 1 mg/kg of body weight per day to approximately 30mg/kg body weight per day.
 3. A method of preventing or treating cancerby administering to a patient a therapeutically effective amount of aflavonoid glycoside or mixtures thereof.
 4. The method of claim 3wherein the therapeutically effective amount of the flavonoid glycosideis approximately 1 mg/kg of body weight per day to approximately 30mg/kg body weight per day.
 5. The method of claim 1 wherein thesteroidal tetraglycoside is of the molecular formula: C₅₁H₈₆O₂₃(molecular weight of 1066) containing open spiro ring Tigogeninaglycone.
 6. The method of claim 1 wherein the steroidal pentaglycosideis of the molecular formula: C₅₇H₉₄O₂₈ (molecular weight of 1226)containing Gitogenin aglycone.
 7. The method of claim 1 wherein thesteroidal pentaglycoside is of the molecular formula: C₅₇H₉₆O₂₉(molecular weight of 1244) containing open spiro ring Gitogeninaglycone.
 8. The method of claim 1 wherein the steroidal hexaglycosideis of the molecular formula: C₆₂H₁₀₄O₃₂ (molecular weight of 1360)containing open spiro ring Tigogenin aglycone.
 9. The method of claim 1wherein the steroidal hexaglycoside is of the molecular formula:C₆₂H₁₀₄O₃₃ (molecular weight of 1376) containing open spiro ringGitogenin aglycone.
 10. The method of claim 1 wherein the steroidalhexaglycoside is of the molecular formula: C₆₃H₁₀₆O₃₃ (molecular weightof 1390) containing open spiro ring Tigogenin aglycone.
 11. The methodof claim 1 wherein the steroidal hexaglycoside is of the molecularformula: C₆₃H₁₀₆O₃₄ (molecular weight of 1406) containing open spiroring Gitogenin aglycone.
 12. The method of claim 3 wherein the flavonoidtetraglycoside is of the molecular formula: C₃₉H₅₀O₂₄ (molecular weightof 902) containing Kaempferol aglycone.